In recent years it has become increasingly evident that physical cues like mechanical micro-environment and geometry, in addition to bio-chemical factors, plays an important role in regulating cell functionalities. Cancer cells also respond to 2D and 3D matrix stiffness in a complex manner using a coordinated, hierarchical mechano-chemical system composed of adhesion receptors and associated signal transduction membrane proteins, the cytoskeletal architecture, and molecular motors. Mechanosensitivity of different cancer cells in vitro are investigated primarily with immortalized human cancer cell lines or murine derived primary cells, not with primary human cancer cells. Hence, little is known about the mechanosensitivity of primary human colon cancer cells in vitro.
In the first part of this dissertation, an optimized protocol is described that demonstrates the isolation of primary human colon cells from healthy and cancerous surgical human tissue samples. Isolated colon cells are then successfully cultured on soft (2 kPa stiffness) and stiff (10 kPa stiffness) polyacrylamide (PA) hydrogels and rigid polystyrene (~3.6 GPa stiffness) substrates functionalized by an extracellular matrix (fibronectin in this case). Fluorescent microbeads are embedded in soft gels near the cell culture surface, and traction assay is performed to assess cellular contractile stresses. Our findings suggest that both the healthy and tumor cells are mechanosensitive. Their average spread area increased with increase in substrate stiffness, and they displayed actin stress fibers and elongated focal adhesions on rigid polystyrene substrates only. Traction cytometry results on soft gels are the first experimental evidence that primary colon tumor cells can generate augmented traction compared to their healthy counterparts. In addition, the contrast between traction patterns and metastatic staging raises the possibility of introducing a potent biophysical marker of cancer metastasis with other molecular biomarkers.
In the second part, we study the role of cell-cell adhesions on the substrate elasticity mediated metastasis-like phenotype (MLP) of human colon carcinoma (HCT-8) cells. HCT-8 cells on PA gels is an attractive in vitro biomaterial platform as they exhibit a dissociative, metastasis-like phenotype (MLP) when cultured on extra-cellular matrix (ECM) coated gels with appropriate mechanical stiffness (20–47 kPa), but not on very stiff (3.6 GPa) polystyrene substrates. We ask the question whether similar morphological transition occurs on cell-cell adhesion molecule, i.e., E-cadherin coated PA gels and if so, how the actin cytoskeleton and focal adhesions compare with ECM mediated response on gels. Experimental results suggest that MLP of HCT-8 cells on PA gels is independent of cell to gel adhesion in 2D in vitro culture.
Finally, we challenge the classical readouts of cellular mechanosensing by examining cell response on soft biological gel, namely, collagen. Our results show that different types of fibroblasts can exhibit spread morphology, well defined actin stress fibers, and larger focal adhesions even on very soft collagen gels (modulus in hundreds of Pascals) as if they are on hard glass substrate (modulus in GPa, several orders of magnitude higher). Strikingly, we show, for the first time, that augmented cell spreading and other hard substrate cytoskeleton architecture on soft collagen gels are not correlated with cell proliferation pattern unlike PA gels and do not require nuclear transcriptional regulator YAP (Yes associated protein) localization in cell nucleus. HCT-8 cell clusters also show augmented spreading/wetting on soft collagen gels unlike PA gels and eventually form confluent monolayer as on rigid glass substrates and MLP is completely inhibited on soft collagen gels. Overall, these results in the third part suggest that cell-material interaction (soft collagen gels in this case) can induce cellular phenotype and cytoskeleton organization in a remarkably distinct manner compared to a classical synthetic polyacrylamide (PA) hydrogel cell culture model and may contribute in designing new functional biomaterials.